Among the challenges that living systems encounter, few are as basic as the requirement that the acidity of both the intracellular and extracellular fluid compartments be maintained within narrow physiological limits. One important, and as yet only partially answered, question is: how do pH-sensing cells detect hyperacidity in the various fluid compartments being monitored? More specifically for taste receptor cells the question becomes: what is being detected and what are the cellular events resulting in excitation of the taste afferent nerves? It would be reasonable to assume that taste receptor cells would monitor the pH of potential foods or beverages (the extracellular pH). Surprising as it might seem that does not appear to be true. We have established that the proximal stimulus for sour response is the intracellular pH. This means that an acid must first enter the taste receptor cell before it can be detected. A major aim of this proposal is to research the various possible ways acids ma enter taste receptor cells. These include diffusion across cell membranes as neutral molecules (e.g. acetic acid), as gases (e.g. carbon dioxide), by electrodiffusion of hydrogen ions, and on special transporters (e.g. monocarboxylate transporters). The sensing cells themselves can only function within a narrow range of intracellular pH values, so a second issue is the determination of the pH regulatory mechanisms present in taste receptor cells and how their function may vary with intracellular pH. It is highly probable that a type 3 sodium-hydrogen exchanger is an important pH regulator in taste cells. This will be ascertained and wider probes initiated. Transduction ultimately depolarizes the taste cells and changes in intracellular calcium can be expected. These also will be probed. We will utilize two basic methods to carry out these studies. We will measure changes in intracellular pH, intracellular calcium, and membrane potential in the taste cells using fluorescent imaging method in a single fungiform papilla with epithelial tissue polarity preserved under voltage clamp conditions. These studies will be complemented by recordings from the chorda tympani with the lingual receptive field under voltage clamp. We have also observed that various bitter tasting substances cause the intracellular pH to become more alkaline. Preliminary studies show that this alkalinity is significantly reduced in the presence of GDP about S, a G-protein blocker. We will test the hypothesis that during bitter taste transduction taste cells become alkaline. For tastants that are not themselves bases (e.g. denatonium) w hypothesize that alkalinization occurs through the activation of a sodium-hydrogen exchanger or other pH regulatory mechanisms. We will test the hypothesis that blockers of pH regulatory mechanisms will also block increases in denatonium induced intracellular calcium. We have shown that the chorda tympani response to denatonium is voltage sensitive indicating that transduction involves modulation of a conductance. Having established a key role for intracellular pH in acid-sensing, these studies involving bitter-tasting compounds will determine if intracellular pH is also a key intermediate in bitter-taste transduction.